In this study, we performed gene expression profiling analysis in RV-infected, well differentiated primary human TBE cells in vitro. We have identified 48 cellular genes that were significantly elevated by both RV16 and RV1B. The results were further confirmed by real-time RT-PCR. The genes induced by infection with either the major group (RV16) or the minor group (RV1B) of RV were quite similar.
We found that primary TBE cells appeared to be resistant to RV infection. We observed very low infectivity of RV16 (< 5%) on well differentiated primary TBE cells at an MOI of 10; in contrast, cell lines such, as HBE1 or BEAS-2B, have much higher susceptibility (~ 100%) at a similar MOI. Consistent with this finding, a recent article reported by our colleague Lopez-Souza and coworkers (23
) demonstrated that well differentiated cells have low susceptibility to RV infection in comparison with the poorly differentiated cells.
Despite the low RV infectivity in our system, we have identified 48 cellular genes that were highly elevated by RV infection. Although many of those genes have been reported previously in other viral infections, such as respiratory syncytial virus (RSV), HIV, etc., they have never been identified before as RV-inducible genes in human airway epithelial cells. Most RV-inducible genes have direct or indirect antiviral activity, as described in Results. The general theme of these RV-inducible genes is to control the viral infection by: (1) inhibition of viral production by attacking multiple steps in the viral life cycles (MX1, OAS1, viperin); (2) enhancement of apoptosis of the infected cells (PKR); (3) secretion of chemokines (CXCL10 and CXCL11) that facilitate the recruitment of cytolytic T cells to remove the infected cells.
Because many RV-induced genes are related in terms of IFN inducibility and antiviral function, we have asked whether a common pathway is responsible for RV-induced gene expression. Indeed, based on the studies using neutralizing antibody and specific chemical inhibitors, we found that both a dsRNA-PKR–dependent pathway and its induced IFN-β autocrine/paracrine–mediated JAK-STAT pathway are involved in the RV-induced gene expression ().
Figure 10. Diagram summarizing the signaling transduction pathways involved in RV-induced gene expression in primary human TBE cells infected with RV16. The replication of viral RNA leads to the formation of dsRNA. dsRNA activates PKR, which is responsible for the (more ...)
Based on the blocking studies, the dsRNA-PKR pathway appears to be the major signaling pathway induced by RV infection. We have also found that dsRNA per se
could robustly elevate similar gene expression in the absence of real viral infection. It is unclear as to how much dsRNA must be generated in the RV-infected cells to exert gene induction; likewise, it is also unclear how much synthetic dsRNA (e.g., polyIC) actually gets into the cells to initiate signaling. Another study (33
) indicated that a much lower dose of dsRNA would be needed if it was transfected into, rather than directly applied onto, the cells. This supports the notion that very few copies of dsRNA are actually required to initiate PKR signaling. It is interesting to note that all the asthma-exacerbating viruses (such as RV, respiratory syncytial virus (34
), and influenza) generate dsRNA in their life cycles. Therefore, it would be fruitful to continue the study of the role of dsRNA in airway inflammatory responses and asthma exacerbations.
We have also shown that RV infection can induce IFN-β secretion, which appears to have a partial effect on the RV-induced gene expression. This finding is consistent with the long-held belief that the dsRNA-PKR pathway can activate IFN production in many other cell types (27
). Interestingly, one most recent study (18
) has demonstrated the causal linkage between an impairment of RV-induced IFN-β production in asthmatic airway epithelial cells and an increase in RV production from those cell cultures. Their finding is consistent with our result regarding the IFN-β effect on antiviral gene expression. However, we did not observe the induction of IFN-β mRNA in the Genechip assays, despite the presence of the corresponding probe sets on the chips. Thus, RV might induce IFN-β secretion without significant elevation of its transcription. Or, the IFN-β probe sets on the chips were just not appropriate for this detection; and the latter emphasizes the importance of using other molecular and biochemical methods to corroborate and complement the Genechip study, as we did in this study. Although the autocrined/paracrined IFN-β had only a partial effect on RV-induced epithelial gene expression, it might also act on other cell types in vivo
. In addition, our results have shown that several key IFN signal molecules (STAT1A, ISGF3G, and IRF7) were highly elevated, which may indicate the high alert status of the IFN system in RV-infected cells. Because many cell types (e.g., macrophages, T lymphocytes, etc.) in the airway are capable of secreting a large amount of IFN, the RV-infected epithelia may manifest a much more robust IFN response with the exogenous IFN challenge in vivo
. Is it possible that the overzealous epithelial IFN response (or antiviral response) is involved in asthma exacerbation? Two recent studies from Holtzman and colleagues supported this notion. Using human asthmatic tissue samples, they have demonstrated that STAT1, a key IFN signaling molecule, is highly elevated and activated in the asthmatic airways (35
). The second study, using a Sendai-virus infection mouse model, further demonstrated that viral infection itself can cause many aspects of asthmatic symptoms (36
Because RV-induced genes were mostly related to antiviral response, and the PKR–IFN-β–JAK–STAT pathway was responsible for their induction, we further tested whether the disruption of this pathway could affect viral production. As expected, both PKR inhibitor (2-AP) and pan-JAK inhibitor (JAK inhibitor I), which had inhibitory effects on antiviral gene expression, significantly enhanced viral production. Interestingly, a recent study has demonstrated that, because of a deficiency of epithelial antiviral response, asthmatic airway epithelial cells could produce much more virus than normal cells upon RV infection (18
). Thus, alteration of epithelial antiviral defense in the diseased condition might contribute to the pathogenesis of airway disease exacerbations.
To our surprise, we did not find any robust elevations of the previously reported cytokine genes, such as IL-6 and IL-8 (12
). This is not due to the lack of sensitivity of the Genechip technique, because subsequent ELISA analysis showed at most a marginal and inconsistent elevation of IL-6 and IL-8 after RV infection (data not sown). We examined the idea that expression of IL-6 and IL-8 is a function of the proportion of cells infected by varying the dose RV in the inoculum. When we increased the infecting dose to an MOI of 100, more than 70% of the cells were infected, and IL-6 and IL-8 expression was significantly induced. However, to achieve an MOI of 100, we needed to use the virus stock at 108
PFU/well, a level that may never occur physiologically, and that presents a daunting challenge to the researcher. Thus, even though the cytokines are significantly elevated in airway secretions from patients with community-acquired or experimentally-induced RV infections, whether they are actually produced by epithelial cells under these conditions remains to be determined.
In summary, we have reported the first gene expression profile analysis in well differentiated human airway epithelial cells after RV infection in a well controlled in vitro environment. We have demonstrated two interacting signaling pathways involved in RV-induced gene expression. Deactivation of these pathways can significantly increase epithelial viral production. This new information will significantly advance our understanding of the pathogenesis of RV infection. Full understanding of the RV-induced airway epithelial response is the first key step to uncovering the pathogenesis of the RV-induced common cold and asthma exacerbations. Because of the broad disease prevalence and the high financial burden, these studies have the potential for great impact on the human health.